Cryptococcus neoformans is a major cause of morbidity and mortality in immunocompromised patients, especially in those afflicted with HIV/AIDS. The overall objective of this proposal is to test the hypothesis that NOT1 is a regulator of stress tolerance and virulence in C. neoformans. A homolog of S. cerevisiae NOT1, a global regulator of nutrient- and stress-sensitive transcription, was identified as an overexpressed transcript in the avirulent, stress-sensitive VAD1 mutant. Specific Aim 1 will define the role of NOT1 mRNA accumulation in stress tolerance and laccase expression in the delta vadl mutant and in wild type C. neoformans. This will be accomplished by profiling NOT1 expression in response to stress using northern blot, assessing the phenotypic consequences of NOT1 overexpression on the wild type, and assessing the effects of NOT1 mRNA suppression in the delta vadl mutant. Specific Aim 2 will determine the role of mRNh synthesis and degradation in the regulation of NOT1 mRNA abundance by separating the two processes experimentally, and define cis elements in the 5' and 3' flanking regions that influence its regulation. Exploring the NOT1 dependent stress response may reveal novel virulence factors in C. neoformans.